Non-invasive blood based monitoring of genomic alterations in cancer

Abstract

The invention provides methods to monitor cell free nucleic acids. The method comprises obtaining a plasma sample from a subject known to have a cancer characterized by a pair of mutually exclusive mutations specific to the cancer; isolating cell free nucleic acids from the plasma sample obtained from the subject; measuring the amount a housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the plasma sample to confirm that the amount of housekeeping gene and/or total DNA in the sample is within a selected range; measuring the amount of a first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the plasma sample; and indicating in a report that the subject has the first mutation when (a) the amount of the housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the plasma sample is within the selected range and (b) the amount of the first mutation is increased as compared to a control amount, wherein the control amount is determined by measuring the apparent amount of the first mutation in control cell free nucleic acids isolated from plasma samples obtained from control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer using measuring conditions substantially the same as those used to measure the amount of the first mutation in the cell free nucleic acids isolated from the plasma sample from the subject.

What is claimed is: 
     
         1 . A method to monitor cell free DNA comprising:
 (i) obtaining a body fluid sample from a subject known to have a cancer characterized by a pair of mutually exclusive mutations specific to the cancer;   (ii) isolating cell free nucleic acids from the body fluid sample obtained from the subject;   (iii) measuring the amount of a housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the body fluid sample, wherein the amount of a housekeeping gene and/or total DNA in the sample correlates to the amount of Line 1 and the amount of Line 1 is within a range of 3,000-700,000 pg/μL;   (iv) measuring the amount of a first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample; and   (v) indicating in a report that the subject has the first of the pair of mutually exclusive mutations when (a) the amount of the housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the body fluid sample correlates to the amount of Line 1 and the amount of Line 1 is within the range of 3,000-700,000 pg/μL and (b) the amount of the first of the pair of mutually exclusive mutations is increased as compared to a control amount,   
       wherein the control amount is the apparent amount of the first of the pair of mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer using measuring conditions substantially the same as those used to measure the amount of the first of the pair of mutually exclusive mutations in the cell free nucleic acids isolated from the body fluid sample from the subject. 
     
     
         2 . The method of  claim 1 , wherein the measuring of: the first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample obtained from the subject is performed by quantitative PCR, microarrays, Next-generation sequencing, chemiluminescence methods, fluorescent methods, digital detection, or mass spectrometry (MALDI-TOF). 
     
     
         3 . The method of  claim 1 , wherein the cancer is lung cancer or colon cancer. 
     
     
         4 . The method of  claim 3 , wherein the pair of mutually exclusive mutations is an epidermal growth factor receptor (EGFR) mutation and a Rat sarcoma (RAS) mutation, wherein the pair of mutually exclusive mutations comprises an epidermal growth factor receptor (EGFR) mutation and a v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, or wherein the pair of mutually exclusive mutations comprises a v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and a Rat sarcoma (RAS) mutation. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 4 , wherein the EGFR mutation is selected from the group consisting of: L858R, T790M, L861Q, G719S, del 19 and exon 20 insertions. 
     
     
         7 . The method of  claim 4 , wherein the KRAS mutation is G12C. 
     
     
         8 - 14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein the amount of the first of the pair of mutually exclusive mutations specific to the cancer is measured by digital droplet PCR. 
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 1 , wherein the amount of the first of the pair of mutually exclusive mutations is measured before and after administration of an anti-cancer therapy to the subject. 
     
     
         18 . The method of  claim 1 , wherein step (i)-step (iv) are repeated so as to monitor the subject's amount of the first of the pair of mutually exclusive mutations over time. 
     
     
         19 . The method of  claim 18 , wherein a decrease in amount of the first of the pair of mutually exclusive mutations indicates that the cancer is stabilizing or decreasing, or wherein an increase in amount of the first of the pair of mutually exclusive mutations indicates that the cancer is increasing. 
     
     
         20 - 22 . (canceled) 
     
     
         23 . A method to monitor cell free DNA comprising:
 (i) obtaining a body fluid sample from a subject known to have a cancer characterized by a pair of mutually exclusive mutations specific to the cancer;   (ii) isolating cell free nucleic acids from the body fluid sample obtained from the subject;   (iii) measuring the amount of a housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the body fluid sample, wherein the amount of the housekeeping gene and/or total DNA in the sample correlates to the amount of Line 1 and the amount of Line 1 is within a range of 3,000-700,000 pg/μL;   (iv) measuring the amount of a first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample; and   (v) measuring the apparent amount of the first of the pair of mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer using measuring conditions substantially the same as those used to measure the amount of the first of the pair of mutually exclusive mutations in the cell free nucleic acids isolated from the body fluid sample from the subject; and   (vi) indicating in a report that the subject has the first of the pair of mutually exclusive mutations when (a) the amount of the housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the body fluid sample correlates to the amount of Line 1 and the amount of Line 1 is within the range of 3,000-700,000 pg/μL and (b) the amount of the first of the pair of mutually exclusive mutations is increased as compared to the apparent amount of the first of the pair of mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from control subjects.   
     
     
         24 . (canceled) 
     
     
         25 . A method to treat cancer comprising:
 (i) obtaining a body fluid sample from a subject known to have a cancer characterized by a pair of mutually exclusive mutations specific to the cancer;   (ii) isolating cell free nucleic acids from the body fluid sample obtained from the subject;   (iii) measuring the amount of a housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the body fluid sample, wherein the amount of housekeeping gene and/or total DNA in the sample correlates to the amount of Line 1 and the amount of Line 1 is within a range of 3,000-700,000 pg/μL;   (iv) measuring the amount of a first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample;   (v) measuring the apparent amount of the first of the mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer using measuring conditions substantially the same as those used to measure the amount of the first of the mutually exclusive mutations in the cell free nucleic acids isolated from the body fluid sample from the subject; and   (vi) treating the subject with an anti-cancer therapy when (a) the amount of the housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the body fluid sample is within the range of 3,000-700,000 pg/μL and (b) the amount of the first of the pair of mutually exclusive mutations is increased as compared to the apparent amount of the first of the pair of mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from control subjects.   
     
     
         26 - 41 . (canceled) 
     
     
         42 . The method of  claim 25 , wherein step (i)-step (iv) are repeated so as to monitor the subject's amount of the first of the pair of mutually exclusive mutations over time. 
     
     
         43 . The method of  claim 42 , wherein administration of the anti-cancer therapy is maintained when the amount of the mutation decreases over time, or wherein the anti-cancer therapy is administered at a higher dosage or is changed when the amount of the mutation increases over time. 
     
     
         44 - 46 . (canceled) 
     
     
         47 . A method comprising:
 (i) administering an anti-cancer therapy to a subject known to have a cancer characterized by a pair of mutually exclusive mutations specific to the cancer;   (ii) obtaining a body fluid sample from the subject;   (iii) isolating cell free nucleic acids from the body fluid sample obtained from the subject;   (iv) measuring the amount of a housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the body fluid sample to confirm that the amount of the housekeeping gene and/or total DNA in the sample correlates to the amount of Line and the amount of Line 1 is within a range of 3,000-700,000 pg/μL;   (v) measuring the amount of a first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample; and   (vi) measuring the apparent amount of the first of the pair of mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer using measuring conditions substantially the same as those used to measure the amount of the first of the pair of mutually exclusive mutations in the cell free nucleic acids isolated from the body fluid sample from the subject.   
     
     
         48 - 68 . (canceled) 
     
     
         69 . The method of  claim 15 , wherein the amount of the first of the pair of mutually exclusive mutations specific to the cancer is determined by:
 preparing at least 2 serial dilutions of the cell free nucleic acids isolated from the body fluid sample;   measuring the amount of the first of the pair of mutually exclusive mutations in the at least 2 serial dilutions using digital droplet PCR; and   evaluating linearity of the measured dilutions to confirm accuracy of the method.   
     
     
         70 . The method of  claim 18 , wherein an increase in amount of the mutation indicates that the cancer is increasing. 
     
     
         71 . The method of  claim 18 , wherein the subject's amount of the first of the pair of mutually exclusive mutations is measured: (a) in a first sample obtained from the subject before the subject received an anti-cancer therapy; and (b) in a second sample obtained from the subject after the subject received an anti-cancer therapy. 
     
     
         72 . The method of  claim 23 , wherein the measuring of: (a) the first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample obtained from the subject and (b) the apparent amount of the first of the pair of mutually exclusive mutations in cell free nucleic acids isolated from control body fluid samples obtained from control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer is performed by microarrays, Next-generation sequencing, chemiluminescence methods, fluorescent methods, digital detection, or mass spectrometry (MALDI-TOF). 
     
     
         73 . A method to monitor cell free DNA comprising:
 (i) obtaining a body fluid sample from a subject known to have a cancer characterized by a pair of mutually exclusive mutations specific to the cancer;   (ii) isolating cell free nucleic acids from the body fluid sample obtained from the subject;   (iii) measuring the amount of a housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the body fluid sample, wherein the amount of a housekeeping gene and/or total DNA in the sample correlates to the amount of Line and the amount of Line 1 is within a range of 3,000-700,000 pg/μL of body fluid;   (iv) measuring the amount of the first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample; and   (v) measuring the apparent amounts of the first of the pair of mutually exclusive mutations in control cell free nucleic acids isolated from body fluid samples obtained from a plurality of control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer using measuring conditions substantially the same as those used to measure the amount of the first of the pair of mutually exclusive mutations in the cell free nucleic acids isolated from the body fluid sample from the subject; and   (vi) indicating a result in a report wherein: the amount measured in (iv) is greater than the highest amount measured in (v) and indicating in a report that the body fluid sample from the subject has the first of the pair of mutually exclusive mutations; or the amount measured in (iv) is less than the highest amount measured in (v) and indicating a report that the body fluid sample from the subject does not have the first of the pair of mutually exclusive mutations.   
     
     
         74 . The method of  claim 73 , wherein the measuring of: the first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the body fluid sample obtained from the subject is performed by quantitative PCR, microarrays, Next-generation sequencing, chemiluminescence methods, fluorescent methods, digital detection, or mass spectrometry (MALDI-TOF). 
     
     
         75 . The method of  claim 73 , wherein the cancer is lung cancer or colon cancer. 
     
     
         76 . The method of  claim 73 , wherein the pair of mutually exclusive mutations is an epidermal growth factor receptor (EGFR) mutation and a Rat sarcoma (RAS) mutation, or wherein the pair of mutually exclusive mutations comprises an epidermal growth factor receptor (EGFR) mutation and a v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, or wherein the pair of mutually exclusive mutations comprises a v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and a Rat sarcoma (RAS) mutation. 
     
     
         77 . The method of  claim 76 , wherein the EGFR mutation is selected from the group consisting of: L858R, T790M, L861Q, G719S, del 19 and exon 20 insertions. 
     
     
         78 . The method of  claim 73 , wherein the amount of the first of the pair of mutually exclusive mutations is measured before and after administration of an anti-cancer therapy to the subject. 
     
     
         79 . The method of  claim 73 , wherein step (i)-step (iv) are repeated so as to monitor the subject's amount of the first of the pair of mutually exclusive mutations over time.